NR3A and NR3B represent a newly identified family of N-methyl-D-aspartate receptor (NMDAR) subunits. In Project III, we will investigate NR3A/3B function at the whole animal/systems level. Our previous data indicate that endogenous NR3 may act as a dominant-interfering molecule of classical NMDAR activity (classical NMDAR refers to receptors composed of NR1/NR2 subunits). Additionally, NR3A/3B can form excitatory glycine-sensitive channels (not responsive to glutamate) in recombinant systems when co-expressed with NR1 in the absence of NR2 subunits. NMDARs are thought to play crucial roles in both normal development and neurotoxicity mediated by glutamate (excitotoxicity). Excitotoxicity has been implicated in neuronal injury and death in focal cerebral hypoxia/ischemia as well as a number of neurodegenerative disorders. The elucidation of the role of NR3A/3B in these events requires animal models. Toward this goal, we have generated four different mutant mice in this project: knockout (KO) mice of NR3A and NR3B, and transgenic (Tg) mice for NR3A and NR3B. Our preliminary studies show that NR3A KO mice exhibit phenotypic changes, including morphological changes (enhanced dendritic spine size and number), electrophysiological alterations (faster rise time of the NMDAR component of the excitatory postsynaptic current [EPSC]), and behavioral modifications. Here, NR3 KO and Tg mice will be subjected to further electrophysiological studies and pathophysiologically relevant experiments to test for in vivo modulation of NMDAR activity by NR3 during neonatal development. In related experiments, this Project identified a gene that is upregulated in NR3A KO mice. The gene, named p16 based on its predicted MW, is a member of a novel and large gene family. Forced expression of p16 in hippocampal neurons increases NMDAR activity, the number of PSD-95 punctae, and the number of dendritic spines. Hence, p16 may be involved in the manifestation of the altered phenotype of NR3A KO mice. The Specific Aims of Project III are ? 1. To examine properties of synaptic transmission and plasticity (long-term potentiation [LTP] and long-term depression [LTD]) in slice preparations from NR3A/3B KO and Tg mice. 2. To examine neuronal damage in vivo following hypoxia/ischemia in NR3A KO and NR3A/3B Tg mice. 3. To examine molecular, functional, and structural properties of p16, the product of a novel gene family that is upregulated in NR3A KO mice.